新硫杆菌亚硫酸盐细胞色素c氧化还原酶的纯化及性质研究

A. Michael Charles , Isamu Suzuki
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No cofactor requirements were demonstrated for the enzyme activity.</p></span></li><li><span>6.</span><span><p>6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO<sub>2</sub> and NH<sub>2</sub>OH did not replace sulfite.</p></span></li><li><span>7.</span><span><p>7. The <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite at pH 8.0 was determined as 4·10<sup>−5</sup> M and 2·10<sup>−5</sup> M with cytochrome <span><math><mtext>c</mtext></math></span> and ferricyanide as electron acceptors, respectively. At pH 6.5 the <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite was 2·10<sup>−6</sup> M with cytochrome <span><math><mtext>c</mtext></math></span>.</p></span></li><li><span>8.</span><span><p>8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The <span><math><mtext>K</mtext><msub><mi></mi><mn>i</mn></msub></math></span> was calculated as 4.5· 10<sup>−3</sup> M.</p></span></li><li><span>9.</span><span><p>9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> and <span><math><mtext>N-</mtext><mtext>ethylmaleimide</mtext></math></span>. The inhibition by <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> was completely reversed by GSH.</p></span></li><li><span>10.</span><span><p>10. Methylene blue, NAD<sup>+</sup>, NADP<sup>+</sup> and O<sub>2</sub> did not replace Fe(CN)<sub>6</sub><sup>3−</sup> or cytochrome <span><math><mtext>c</mtext></math></span> as electron acceptor for sulfite oxidase. <em>T. novellus</em> cytochrome <span><math><mtext>c</mtext></math></span> was reduced by the enzyme and sulfite and subsequently oxidized by <em>T. novellus</em> cytochrome oxidase.</p></span></li><li><span>11.</span><span><p>11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of <em>T. novellus</em>.</p></span></li><li><span>12.</span><span><p>12. 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No cofactor requirements were demonstrated for the enzyme activity.</p></span></li><li><span>6.</span><span><p>6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO<sub>2</sub> and NH<sub>2</sub>OH did not replace sulfite.</p></span></li><li><span>7.</span><span><p>7. The <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite at pH 8.0 was determined as 4·10<sup>−5</sup> M and 2·10<sup>−5</sup> M with cytochrome <span><math><mtext>c</mtext></math></span> and ferricyanide as electron acceptors, respectively. At pH 6.5 the <span><math><mtext>K</mtext><msub><mi></mi><mn>m</mn></msub></math></span> for sulfite was 2·10<sup>−6</sup> M with cytochrome <span><math><mtext>c</mtext></math></span>.</p></span></li><li><span>8.</span><span><p>8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The <span><math><mtext>K</mtext><msub><mi></mi><mn>i</mn></msub></math></span> was calculated as 4.5· 10<sup>−3</sup> M.</p></span></li><li><span>9.</span><span><p>9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> and <span><math><mtext>N-</mtext><mtext>ethylmaleimide</mtext></math></span>. The inhibition by <span><math><mtext>p-</mtext><mtext>hydroxymercuribenzoate</mtext></math></span> was completely reversed by GSH.</p></span></li><li><span>10.</span><span><p>10. Methylene blue, NAD<sup>+</sup>, NADP<sup>+</sup> and O<sub>2</sub> did not replace Fe(CN)<sub>6</sub><sup>3−</sup> or cytochrome <span><math><mtext>c</mtext></math></span> as electron acceptor for sulfite oxidase. <em>T. novellus</em> cytochrome <span><math><mtext>c</mtext></math></span> was reduced by the enzyme and sulfite and subsequently oxidized by <em>T. novellus</em> cytochrome oxidase.</p></span></li><li><span>11.</span><span><p>11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of <em>T. novellus</em>.</p></span></li><li><span>12.</span><span><p>12. 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引用次数: 46

摘要

1.1. 从新硫杆菌中纯化亚硫酸盐氧化酶(亚硫酸盐:细胞色素c氧化还原酶),并对其性质进行了研究。亚硫酸盐氧化酶不需要AMP进行亚硫酸盐氧化,与硫代硫杆菌的硫酸磷酸腺苷还原酶不同。该酶用亚硫酸盐化学计量还原铁氰化物或细胞色素c,每摩尔亚硫酸盐还原2mol电子受体。磷酸钾缓冲液对酶的最适pH值在8左右。酶活性不需要辅助因子。6.6。该酶对亚硫酸盐作为底物具有特异性。硫代硫酸盐、半胱氨酸、谷胱甘肽、NANO2和NH2OH不能替代亚硫酸盐。以细胞色素c和铁氰化物为电子受体,测定了pH 8.0下亚硫酸盐的Km分别为4·10−5 M和2·10−5 M。在pH 6.5时,亚硫酸盐的Km为2·10−6 M,细胞色素c.8.8。各种盐和缓冲液抑制酶活性。与亚硫酸盐相比,NaCl的抑制作用是竞争性的。Ki值为4.5·10−3 M.9.9。对羟基汞苯甲酸酯和n -乙基马来酰亚胺等多种巯基抑制剂对这些酶有强烈的抑制作用。GSH.10.10完全逆转了对羟基汞苯甲酸酯的抑制作用。亚甲基蓝、NAD+、NADP+和O2不能取代Fe(CN)63−或细胞色素c作为亚硫酸盐氧化酶的电子受体。细胞色素c被酶和亚硫酸盐还原,随后被细胞色素氧化酶氧化。11.11。氧化磷酸化与亚硫酸盐氧化偶联,P/O比较低。由下式可知,毛竹亚硫酸盐氧化酶催化亚硫酸盐氧化。SO32−+ 2 cyt c Fe3+ + H2O→SO22−+ 2 cyt c Fe2−+ 2h +
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Purification and properties of sulfite: Cytochrome c oxido-reductase from Thiobacillus novellus

  • 1.

    1. Sulfite oxidase (sulfite: cytochrome c oxidoreductase) was purified from Thiobacillus novellus and the properties were studied.

  • 2.

    2. Sulfite oxidase did not require AMP for sulfite oxidation and was distinct from adenosine phosphosulfate reductase of Thiobacillus thioparus.

  • 3.

    3. The enzyme reduced either ferricyanide or cytochrome c with sulfite stoichiometrically, reducing 2 moles of the electron acceptor for every mole of sulfite.

  • 4.

    4. the pH optimum of enzyme was around 8 with potassium phosphate buffers.

  • 5.

    5. No cofactor requirements were demonstrated for the enzyme activity.

  • 6.

    6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO2 and NH2OH did not replace sulfite.

  • 7.

    7. The Km for sulfite at pH 8.0 was determined as 4·10−5 M and 2·10−5 M with cytochrome c and ferricyanide as electron acceptors, respectively. At pH 6.5 the Km for sulfite was 2·10−6 M with cytochrome c.

  • 8.

    8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The Ki was calculated as 4.5· 10−3 M.

  • 9.

    9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding p-hydroxymercuribenzoate and N-ethylmaleimide. The inhibition by p-hydroxymercuribenzoate was completely reversed by GSH.

  • 10.

    10. Methylene blue, NAD+, NADP+ and O2 did not replace Fe(CN)63− or cytochrome c as electron acceptor for sulfite oxidase. T. novellus cytochrome c was reduced by the enzyme and sulfite and subsequently oxidized by T. novellus cytochrome oxidase.

  • 11.

    11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of T. novellus.

  • 12.

    12. It is concluded that sulfite oxidase of T. novellus catalyzes the oxidation of sulfite according to the following equation. SO32− + 2 cyt c Fe3+ + H2OSO22− + 2 cyt c Fe2− + 2 H+

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